Nunc glass base dish

We performed mitochondria, ER (endoplasmic reticulum) and Golgi detection assays in MI stage oocytes of control and KIF3A-KD (knock down) groups. The oocytes were placed in the M2 medium with Mito-tracker probes (Invitrogen, M7512) and the mitochondrial potential probe 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1, Beyotime, C2005, Nantong, China) reagent for 30 min at 37 °C to detect mitochondrial distribution and mitochondrial membrane potential, respectively. The oocytes were placed in the M2 medium of ER-Tracker Red (Beyotime, C1041) for 30 min at 37 °C to detect ER. After washing three times with M2 medium, oocytes were stained with Hoechst 33,342 for 10 min, then transferred to Nunc glass base dish (Thermo scientific, 150,680). We observed and photographed the samples with a confocal laser scanning microscope (Zeiss, LSM 900 META).

For immunofluorescence staining, MuSCs were cultured on Nunc Glass Base Dish (Thermo, Cat# 150682) and fixed with 4% paraformaldehyde (PFA) (Beyotime, Nanjing, China, Cat# P0099) overnight at 4 °C, then permeabilized with 0.5% Triton X-100 in PBS for 30 min, washed three times with PBS. Samples were incubated overnight at 4 °C with primary antibodies: YAP (1:800, ABclonal, Wuhan, China, CAT#A1002), MYHC (1:500, Abcam, Cambridge, UK, CAT# ab37484), then washed three times, incubated with secondary antibodies: Alexa Fluor 594 goat anti-mouse IgG(H+L) (Invitrogen, Cat# A11005) or Alexa Fluor 488 goat anti-Rabbit IgG(H+L) (Invitrogen, Carlsbad, CA, USA, Cat# A11034) (1:500) for 1 h and then washed three times. Samples were mounted with VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA, Cat# H-1200). Images were obtained using a laser scanning confocal microscope (Leica, TCS SP8 X, Watzlar, Germany).

Phase-contrast and differential interference contrast (DIC) analyses were performed with a Nikon Eclipse Ti2 inverted microscope, with a Nikon DS-Ri2 Camera and a Nikon CFI Plan Apochromat Lambda 100X oil immersion objective (numerical aperture 1.45). For phase contrast analyses the objective additionally contained a Ph3 module. The Images were processed employing the Nikon NIS-Elements software (Version 5.30, https://www.microscope.healthcare.nikon.com/de_EU/products/software/nis-elements) and FIJI (Version 2.9.0, https://downloads.imagej.net/fiji/releases/2.9.0/). Specimens were immobilised on a medium-supplemented 1% (w/v) agarose cushion, for time-lapse analyses the cover glass was sealed with VLAP (33% (w/w) vaseline, 33% (w/w) lanoline, 33% (w/w) paraffin) against the slide to minimise evaporation. Additional time-lapse analyses were performed in a Nunc Glass Base Dish (Thermo Fisher Scientific, 12 mm), which was incubated with supernatant of a growing culture for 24 h. Fluid was then discarded, and remaining cells were immobilised with an agarose cushion as described above.

Embryos were prepared using the previously described approach and mounted on a 27 mm Nunc Glass Base Dish (Thermo Fisher Scientific). Instead of using halocarbon oil, embryos were covered in a drop of room temperature water, after which a silicon tube with a diameter of 1.5 mm was placed in the dish as shown in Figure 5. After the dish was placed on the microscope and embryos found the heat shock was started by the addition of 5 ml of 37 °C water that covered the embryos and filled most of the dish and acted as a thermal bath for the embryos. In order to maintain the water in the dish at 37 °C over the course of ∼ 10 minutes, water flowed continuously through the tube from a water bath at higher temperature than 37 °C. The water that flowed through the tube did not directly contact the water in the dish, but passively transferred thermal energy through the silicon tube to the water in contact with the embryos. Through empirical tests it was established that with the flow rate used, maintaining the temperature of the water bath at 49 °C ensured the water in the dish stayed at a constant 37 °C. To halt the heat shock the source of the flow water was changed to room temperature which decreased the temperature of the water in the dish to room temperature over the course of several minutes.

HaCaT cells (6 × 10⁵/dish) were seeded in a 12-mm Nunc Glass Base dish (Thermo Fisher Scientific). The cells were preincubated with 300 μg/ml of SSWex for 1 h, followed by stimulation with 10 ng/ml IFN-γ/TNF-α for 1 h at 37°C. The stimulated cells were washed twice with PBS and fixed in 3% paraformaldehyde (diluted with PBS) for 20 min at 4°C. The fixed cells were washed four times with 0.1% TritonX-100 buffer for 10 min, blocked with 3% BSA (diluted with 0.1% TritonX-100 buffer) for 1 h at room temperature, and incubated with the primary antibodies (1:500) overnight at 4°C. The primary antibodies used here were the same as those used in Western blot analysis. Subsequently, the cells were incubated with Alexa Fluor 594 or 488 anti-rabbit or mouse IgG secondary antibody (1:500) for 2 h at 4°C. The nuclei were stained using DRAQ5™ in blocking buffer. After incubation of 15 min, the cells were acquired using an FV10i confocal microscope (Olympus, Tokyo, Japan).